Journal of ISHWM Vol 5 Issue 1 April 2006 21

ARTICLE

Effects of Vermicomposting on Microbiological Flora of Infected Biomedical Waste

Umesh B Mathur*, Lalji K Verma**, Jitendra N Srivastava*** *Principal Medical Officer, HQ Central Air Command, Indian Air Force, & Former Senior Adviser & Professor (HOD) Department of Pathology, Air Force Hospital , Bangalore - PI of the project. **Former Director General Medical Services Indian Air Force - Progenitor of the Concept/Hypothesis. ***Former Deputy Director Medical Services Indian Air Force Co-worker in the Project.

ABSTRACT

As an offshoot of WHO aided pilot project on hospital waste management in Bangalore India composting of bio- degradable part of infected hospital waste using earthworms (Eisenia fetida and Eudrilus eugeniae) was conceived and carried out in Command Hospital (CHAFB) .The aim was to study whether vermicomposting was an effective method for rendering infected waste to non-infective, the end product being eco-friendly as well and to assess the efficacy of vermicomposting ,.Results are suggestive of prospects of use of Vermicomposting to convert infected biodegradable waste containing various pathogenic microorganisms to an innocuous waste containing commensals like citrobactor frundii and aerobic spore bearing microorganism usually found in the soil all over. The paper highlights detailed methodology of a systematic attempt towards examining a hitherto less exposed option.

Keywords: Vermi-Composting, Biomedical Waste, Microbiological Flora

Hospital wastes are hazardous and need to be degradable component of hospital waste. disinfected before disposal. One out of eleven WHO aided pilot projects on Hospital Waste Management Vermicomposting refers to production of in India during biennium, 1999-2000 was allotted compost by growing/ breeding earthworms as to the Command Hospital, Air Force, Bangalore these worms in the process of feeding on waste (CHAFB). Project commenced at CHAFB on 10 cause biooxidation by relentless turning, Jan 1999 as Air Mshl (then AVM) Lalji K Verma fragmentation and aeration of waste by devouring as principal investigator to lay down a proper resulting in homogeneous and stabilized humus hospital waste management system (Verma and like product which is an ideal nutrient for plants Srivastava, 2000). As an offshoot of this project thus used as manure. Vermicomposting of composting of bio-degradable part of infected biodegradable Municipal Solid Waste & household hospital waste using earthworms (Eisenia fetida waste is in vogue in many places and instances and Eudrilus eugeniae) was conceived and carried but there is no available literature on use of out. It was conceived that bio-degradable vermicomposting technique for treatment and component of the waste could be disinfected with disposal of infected bio medical waste. The aim was process of vermicomposting without causing any to see whether vermicomposting was an effective adverse impact on the environment & human method for rendering infected waste to non- health. Vermi composting as an effective method infective, the end product being eco-friendly as to treat bio-degradable municipal or household well. To assess the efficacy of vermicomposting in waste is an age old proven methodology. The same this regard microbiological cultures (aerobic concept was applied for treating infected bio- cultures) of vermicompost were carried out. 22 Journal of ISHWM Vol 5 Issue 1 April 2006 MATERIAL AND METHODS Preliminary studies were carried out from vermin-composting beds, each measuring 3.5ft.xx3.5ft xx1ft., lined by a single layer of earthen tiles made on a cemented floor in a few rooms having asbestos sheets ceiling. Twelve such beds were used for studies. These beds were charged with infected biodegradable waste comprising of cotton/gauze pieces soiled with blood, pus and body fluids etc. In many cases it would have been admixed with antiseptics/antibiotics used for dressing of wounds etc. It was covered with dry Fig 1: Vermi-compost pit containing leaves after watering and admixing with an infectious waste adequate number of earthworms (Eisenia fetida The details of microorganisms isolated from and Eudrilus eugeniae). They were left various vermicomposting beds (No I to VI) at undisturbed for vermicomposting and compost thus different intervals is shown in Table- I. formed later was subjected to aerobic microbiological cultures at periodic intervals till full Samples from beds VII to XII showed similar composting took place (Photograph I). findings. Preliminary studies of initial samples

Table - I

Vermi- Microorganism isolated from vermicompost of different duration

composting Beds 3 Wk. 6 Wk. 9 Wk. 12 Wk. 15 Wk. 18Wks Bed No I Esch coli Esch coli Esch coli Citrobacter Citrobacter sp Citrobacter Staph Aerobic Aerobic sp Aerobic Aerobic sp Aerobic aureus spore spore spore spore spore Bed No II Esch coli Esch coli Esch coli Citrobacter Citrobacter Citrobacter sp Staph Staph Citrobacter sp sp aureus aureus sp Citrobacter sp Bed No III Aerobic Aerobic Esch coli Citrobacter Citrobacter Citrobacter spaore spaore Proteus sp Aerobic spAerobic sp Aerobic Aerobic spore spore spore spore Bed No IV No microor No microo Citrobacter Citrobacter Citrobacter Citrobacter ganism rganism spAerobic spAerobic spAerobic sp Aerobic Isolated spore spore Spore spore Bed No V Staph Proteus Proteus Citrobacter Citrobacter Citrobacter aureus Esch coli Citrobacter spAerobic spAerobic spAerobic Esch coli Pseudomo sp Aerobic spore spore spore Pseudomo nas spore nas Bed No VI Esch coli Esch coli Esch coli Pseudomon Citrobacter Citrobacter Proteus Pseudomo Pseudomon as Citrobact sp sp nas Proteus as Proteus er sp Aerobic spore Journal of ISHWM Vol 5 Issue 1 April 2006 23 from different vermicomposting beds showed intestine, and excreted in very large number in growth of different bacterial flora on aerobic faeces. It can contaminate the environment, culture. Microorganisms isolated were Esch coli including the soil widely, and bacilli may survive and Staph aureus, Esch coli and aerobic spore without growth for several days to a few weeks bearing microorganism, Esch coli and Proteus sp, outside the body. When it is found in a water Pseudomonas sp along with aerobic spore bearing supply, it indicates that the supply has recently microorganism and Citrobacter sp while samples been subjected to contamination with human or from some vermicomposting beds had an animal faeces. admixture of all these pathogens. In a few beds initially no microorganism was isolated and this Following vermin-composting, it was observed could have been due to effects of antiseptics/ that instead of Esch coli, Citrobacter frundii and antibiotics in the compost sample collected for aerobic spore bearing microorganisms were culture. As the vermicomposting process advanced isolated. Pathogens such as Staph aureus, Proteus it was observed that mainly Citrobacter sp was and Pseudomonas sp were not isolated from the isolated (except in a few cases where aerobic spore samples. Citrobacter frundii was isolated on bearing microorganism and Citrobacter sp. were repeated cultures after vermicomposting isolated) from later samples. It is interesting to irrespective of duration of vermicomposting, from note that even in beds where initially no 3 weeks to 18 weeks. Incidentally Citrobacter is microorganism was isolated, Citrobacter sp were one of the coliform bacteria and it commonly grows isolated in later samples with continued in soil, vegetation, and water outside the body. vermicomposting process. As infected Aerobic spore bearing microorganisms were also biodegradable waste was used for vermin- isolated in the compost. These organisms are composting, initial cultures from immature compost usually found in atmosphere and are non- showed isolation of Esch Coli, Staph aureus, pathogens. Pseudomonas sp and Proteus sp. This was as Findings as described above during the expected as these are usual pathogens found in preliminary studies required further studies. It was purulent material. Strains of Esch coli and related therefore decided to conduct the study under strict coliform bacteria are predominant among the controlled environmental conditions for drawing aerobic commensals flora present in the gut of any conclusion. It was necessary that cultures human beings and animals and are also widely were done on samples of vermicomposting which distributed in the environment. All persons have has been processed under controls. a rich flora of Esch coli in the lower ileum and in the colon. Esch coli is incriminated as a pathogen A research project was undertaken with an outside the gut and particularly in the urinary aim to find out the effect of vermicomposting on tract and in wounds where the infection may be microbiological flora of infected biodegradable endogenous or acquired from exogenous source. It biomedical waste by carrying out microbiological grows in human beings and animals mainly in cultures (aerobic cultures) of infected biomedical waste at the beginning of vermicomposting process, and at intervals of three weeks till maturity of compost. The protocol adopted for microbiological cultures (aerobic cultures) in this study were strictly followed to avoid variations due to extraneous physical factors. Since the preliminary studies revealed growth of Citrobacter bacteria on the culture of vermicompost, special care was taken to avoid contamination of vermicompost sites with refuse and dust generated at time of cleaning and sweeping. Besides material used for making bed for earthworms, dried leaves and water used for vermicomposting throughout was checked for coliform microorganisms to ensure Fig 2: Treated waste containing worms that it is free of all microorganisms. 24 Journal of ISHWM Vol 5 Issue 1 April 2006

To prevent contamination of beds compost in sterile wide mouthed tubes. The vermicompost of infected biodegradable waste was complete specimen was inoculated in a fluid carried out in fabricated metallic vermicomposting culture media i.e. Nutrient broth and incubated at containers, each measuring 3 ft x1.5 ft x 1.5 ft 37 degree C aerobically for 16-24 hours. Fluid having a sliding lid duly perforated for watering culture media was used as bacteria may not grow and dissipation of heat generated during the on solid media if only a few viable organisms are composting. Besides, the containers had a few present in the specimen, or if the material small perforations, each measuring 3 mm in submitted contains substances that inhibit diameter at the base and sides to prevent bacterial growth (e.g. antibiotics). Inoculation of accumulation of toxic nitrogenous waste but small a fluid medium however not only ensures rapid enough to prevent entry of rodents/reptiles. These multiplication of small number of organisms but containers were labeled and kept on metallic also dilutes the concentration of inhibitory stands. Legs of the stand were kept inside bowls substances allowing growth to take place. Growth containing water duly covered with a layer of in fluid media was investigated by sub culturing kerosene oil so as to prevent entry of crawling on solid media i.e. MacConkey medium, blood agar insects into the vermicomposting containers. etc for isolation and later identification of Three containers were prepared as follows: - microorganisms by biochemical tests. Similar samples were taken from the negative control 1. Test Container container and subjected to similar biological culture 2. Positive Control Container studies at similar intervals. 3. Negative Control Container RESULTS Each container was duly prepared after The details of microorganisms isolated from coating and maturation of earthworm beds (made compost obtained during vermicomposting from up of coconut husk & shredded papers weighing test, positive and negative control containers at approximately 14 Kg). The Test container was different intervals is shown in Table - II. thereafter charged with 6.5 kg of infected biodegradable waste generated in various wards/ The microbiological cultures (aerobic cultures) departments, comprising of waste soiled with of compost obtained after vermicomposting from blood, pus and body fluids, and admixed with 750 test container revealed the growth of Esch coli, gm of earthworms (Eisinia foetida and Eudrilus Proteus vulgaris, Staph aureus, Pseudomonas eugeniae) for composting. The Positive Control pyocyaneae and aerobic spore bearing Container was charged with 6.5 Kg of waste microorganism initially by repeated cultures till prepared in biological laboratory containing nine weeks of vermicomposting. Later tests biodegradable waste prepared by soaking cotton/ revealed that above mentioned pathogenic gauze swabs with blood containing microbes in microorganisms were not isolated and Citrobactor large numbers viz. Esch coli, Pseudomonas frundii and aerobic spore bearing microorganisms pyocyaneae and Staph aureus, and similarly only were isolated. Microbiological cultures of admixed with 750 gm of earthworms (Eisinia compost generated in container used as a positive foetida and Eudrilus eugeniae). Vermi-composting control revealed the growth of Staph aureus up was allowed to proceed under observation. The to three weeks of vermicomposting while Esch coli compost thus formed in these two containers was and Pseudomonas pyocyaneae could be isolated up subjected to microbiological cultures (aerobic to nine weeks and twelve weeks respectively. From cultures) at interval of three weeks till composting ninth week Citrobactor frundii and aerobic spore process was complete, which was 18 weeks. bearing microbes were also isolated but beyond Samples from the Negative Control Container (in twelve weeks, Citrobacter frundii and aerobic spore which worms were not admixed) were also bearing microbes only were isolated. similarly tested periodically for biological culture. Examination of infected biodegradable waste charged in container used as negative control Specimens (appr 15-20 gm) of compost after revealed no composting of waste in the absence of vermicomposting from test & positive control earthworms till eighteen weeks of study. The waste containers were collected from deeper portion of appeared decayed but still discernible, as cotton Journal of ISHWM Vol 5 Issue 1 April 2006 25

Table - II

Vermicomp osting Microorganism Isolated from vermicompost of different duration Containers 3 Wks 6 Wks 9 Wks 12 Wks 15 Wks 18 Wks Test Esch coli Esch coli Esch coli Citrobactor Citrobac Citrobactor Container Proteus Proteus Citrobac Aerobic Spore tor Aerobic Spore vulgaris vulgaris tor Aerobic Staph Staph aureus Aerobic Spore aureus Spore Positive Esch Coli Pseudomonas Pseudom Pseudomonas Citrobac Citrobactor Control Staph pyocyaneae onas pyocyaneae tor frundii container aureus pyocyane Citrobactor frundii Aerobic Spore Pseudomona ae frundii Aerobic Aerobic s pyocyaneae Citrobac Spore Spore tor frundii Aerobic Spore Negative Esch coli Esch coli Esch coli Esch coli Proteus Esch coli Esch coli Control Staph Staph aureus Staph vulgaris Staph Proteus Proteus container aureus Pseudomonas aureus aureus Aerobic vulgaris vulgaris Pseudomona pyocyaneae Aerobic Spore Aerobic Aerobic Spore s pyocyaneae Klebsiella Spore Spore Klebsiella aerogenes aerogenes

Similar findings were obtained by repeated cultures carried out later on. fibers could be identified. Microbiological cultures be used as plant nutrient. of specimens from negative control vermicomposting container revealed growth of a b. The pathogenic microorganisms present in wide range of pathogenic microorganisms viz. Esch infected biodegradable waste initially viz. Esch coli, Staph aureus, Klebsiella aerogenes, Proteus coli, Staph aureus, Proteus vulgaris and vulgaris and Pseudomonas pyocyaneae till Pseudomonas pyocyaneae were not isolated eighteen weeks of study. Aerobic spore bearing from the compost after twelve weeks of microorganisms could also be isolated after nine vermicomposting. However these pathogens weeks of study. Citrobacter frundii was not isolated were replaced by commensals like Citrobactor from this container on repeated aerobic cultures. frundii and aerobic spore bearing Appearances of Citrobacter frundii in compost microorganisms. after vermicomposting led us to dissect the c. Citrobacter frundii was isolated repeatedly earthworms (Eisinia fetida and Eudrilus eugeniae) from the alimentary canal of the earthworms and to carry out microbiological cultures from its used for vermicomposting. alimentary canal. It revealed pure growth of Citrobacter frundii on repeated cultures form d. Earthworms used for composting hasten the different earthworms. The results obtained process of composting. The earthworms in indicate that:- each container (test & positive control containers) increased from 750 gms to 1.250 a. There is a marked decrease in the net content kg. of waste after vermicomposting and the total content in test as well as in positive control e. There was no appreciable change in container decreased from 20.5 kg to 4.5 kg microbiological content or weight in the each with the formation of compost that could 26 Journal of ISHWM Vol 5 Issue 1 April 2006

negative containercontainer. biomedical waste remarkably with the resultant manure formation that could be used as a plant DISCUSSION nutrient thus economically beneficial for the organization. Dominguez et al (1997) studied the similarities and differences in composting systems CONCLUSION that can be operated in open or in vessel system with or without worms. They found that organic The importance of biomedical waste waste can be broken down and fragmented rapidly management by safe, cheap and easy methods by earthworms, resulting in a stable nontoxic need no further emphasis. Hospital wastes are material with good structure. Their preliminary hazardous thus before it leaves the hospital it research showed that vermicomposting involves should be safe for humans, animals and great reduction in populations of pathogenic environment. Composting as one of the simple, microorganisms. After 60 days of vermicomposting, cheap and easy methods of management of animal fecal coliform bacteria in bio-solids dropped from organic wastes, and has been in vogue since time 39000 MPN/g to0 MPN/g. In that same period, immemorial. This is still a common method of salmonella sp. dropped from <3 MPN/g to <1 generating manure all over the world. MPN/g. They stated that as an aerobic process, Vermicomposting is more efficient method of composting lead to a nitrogen mineralization and management of waste where in biooxidation and the use of earthworms in vermicomposting stabilization of organic matter is carried out with increases and accelerate this nitrogen the help of a breed of earthworms (Eisenia fetida mineralization rate. The humidification processes and Eudrilus eugenia) which are sturdy, have that take place during the maturation stage of tremendous appetite and reproduce at a fast rate. composting are greater and faster during It has been found that besides resulting in marked vermicomposting. It also decreases heavy metals decrease in the net content of waste, and the final product may contain hormone like vermicomposting converts the infected compounds that accelerate plant growth. Our biodegradable waste containing various pathogenic research project revealed that human pathogens microorganisms to an innocuous waste containing did not survive vermicomposting and commensals like citrobactor frundii and aerobic microorganisms like Staph aureus, Proteus spore bearing microorganism usually found in the vulgaris, Pseudomonas pyocyaneae and Esch coli soil all over. were not isolated after twelve weeks of vermicomposting. However Citrobacter frundii and REFERENCES aerobic spore bearing microorganism were isolated. 1. Verma LK & Srivastav JN (2000) Final Report-WHO Incidentally both are the commensals found in soil Pilot Project and the alimentary canal of earthworms. Besides, 2. Dominguez J Edwards CA and Subler S. A comparison it established that earthworms not only hasten the of vermin-composting and composting. Biocycle, April process of composting but also activate composting 1997, 57 – 5 and helps reducing the net content of infected

Waste that cannot be incinerated

Chlorinated plastics, volatile toxic wastes such as mercury.

Patient-contaminated plastics, non-plastics and infectious laboratory wastes may be treated by steam sterilization in autoclavable bags or microwave treatment. Shredding should follow both these methods. In case of non-availability of the above, chemical treatment with 1% hypochlorite or a similar disinfectant is recommended. However, excessive use of chemical disinfectants may be a health and environmental hazard.

Source: WHO Website